The role of residues T248, Y249 and T422 in the function of human pregnane X receptor

Abstract

The pregnane X receptor (PXR) is a key xenobiotic receptor that regulates the expression of numerous drug-metabolizing enzymes. Some posttranslational mechanisms modulate its transcriptional activity. Although several kinases have been shown to directly phosphorylate this receptor, little is known about phosphorylation sites of PXR. In the present work, we examined T248, Y249 and T422 putative phosphorylation sites determined based on in silico consensus kinase site prediction analysis. T248 and T422 residues are critical for the interaction of the PXR ligand-binding domain and the activation function-2 (AF2) domain. Site-directed mutagenesis analysis was performed to generate phospho-deficient and phospho-mimetic mutants. We examined transactivation activity of the PXR mutants in gene reporter assays, formation of PXRmutant/RXRα heterodimer, binding of PXR mutants to the CYP3A4 gene response element DR3 and CYP3A4 expression in HepG2 cells after expression of the mutants. We found that T248D mutant activated CYP3A4 transactivation constitutively regardless of the presence or absence of a ligand. Contrary, T248V mutant exhibited low basal and ligand-inducible transactivation capacity as compared to wild-type PXR. Dose-response analysis revealed reduced ligand-dependent transactivation potency of PXR Y249D mutant. Transactivation of the CYP3A4 promoter was abolished with T422A/D mutants. All PXR mutants formed heterodimer with RXRα at a similar level to that observed with wild-type PXR. The ability to bind to DNA in vitro was substantially decreased in case of T248D, T422D and T248V mutants. Our data thus indicate that phosphorylation of T248, Y249 and T422 residues may be critical for the both basal and ligand-activated function of PXR.

Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

“…Some of these human PXR SNPs have been associated with change in PXR expression levels [e.g. (Lamba et al, 2008)]; changes in PXR function, including the ability of DNA-binding, response element preference, basal CYP3A expression and the induction potential of the receptor (Doricakova et al, 2013; Hustert et al, 2001; Lamba et al, 2008; Oleson et al, 2010; Zhang et al, 2001); changes in the hepatic clearance of various pharmaceuticals (Schipani et al, 2010); and susceptibility to disease (Andersen et al, 2011; Dring et al, 2006). …”

Acetylation of lysine 109 modulates pregnane X receptor DNA binding and transcriptional activity

“…Procedure was performed as previously described [32]. HepG2 cells (10 6 /well) were transiently transfected employing FuGENE HD reagent (Roche) with 1 µg/well of expression plasmid encoding WT PXR or its mutated forms.…”

Post-translational and Post-transcriptional Modifications of Pregnane X Receptor (PXR) in Regulation of the Cytochrome P450 Superfamily

“…The effects of site-specific phosphorylation of PXR by kinases interfere with a wide variety of its functions involving subcellular localization, dimerization, DNA binding, and co-regulator interaction [38,43–45,47,51]. While phosphorylation generally may contribute to both activation or termination activity in NRs [50], direct phosphorylation in the case of human PXR leads mostly to negative response in its transcriptional activity [9].…”